Chiral nucleic acid adjuvant having anti-allergic activity, and anti-allergic agent

ABSTRACT

An adjuvant which comprises oligonucleotides which comprise two to four CpG motifs each represented by 5′-X1CpGX2-3′ and has a length of 14 to 32 nucleotides, wherein a nucleic acid at 3′ end side of at least two CpG motifs is connected by phosphorothioate linkage, wherein each nucleic acids at 3′ end and 5′ end of the oligonucleotide is S type nucleic acids connected by phosphorothioate linkage, and wherein the oligonucleotide comprises at least one nucleic acid without phosphorothioate modification. The present invention relates to an adjuvant comprising the oligonucleotide represented by the sequence number 67. The present invention relates to an anti-allergic agent comprising these adjuvant.

FIELD OF THE INVENTION

This invention is directed to a chiral nucleic acid adjuvant and ananti-allergic agent that have anti-allergic effect by means of CpGoligonucleotides. In more detail, this invention is directed to thechiral nucleic acid adjuvant and the like that has CpG oligonucleotideshaving PS and PO structures.

BACKGROUND OF THE INVENTION

JP 2002-513763 T (Patent Literature 1), JP 2002-154397 A (PatentLiterature 2), and JP 2002-521489 T (Patent Literature 3) disclose CpGoligonucleotides and the method of manufacturing them.

JP 2010-504750 T (Patent Literature 4) discloses that oligonucleotides,which have lipophilic substituted nucleotide analogues out of CpG motif,cause production of interferon-α (IFN-α).

Non Patent Literature 1 discloses that the S-form stereoisomer of CpGoligonucleotide trimer promotes MAPK signal.

Non Patent Literature 2 discloses PF-3512676 (Sequence No. 119), allparts of the sequence are phosphorothioated and S-form stereoisomer.Natural oligonucleic acid is readily reduced in vivo. On the other hand,oligonucleic acid with P—S modifications is difficult to be reduced invivo. In the oligonucleic acid with P—S modifications phosphoric acidester bond of oligo nucleic acid (P—O bond) is replaced with phosphoricacid thioester bond (P—S bond).

The Non Patent Literature 3 discloses that the CpG oligonucleotide(oligonucleotide having a CpG sequence) activates Th1 immune paththrough the Toll-like receptor (TLR9). CpG oligonucleotides can beclassified into three types: class A-C. In CpG oligonucleotidesclassified as class A, the 3′ and 5′-phosphate-binding site of the endof 1-4 bases are phosphorothioate linkages (PS-binding), shows a strongIFN-α production inducing ability. However, its effects on B cellproliferation are weak. On the other hand, CpG oligonucleotides areclassified as class B and C, all the phosphate binding sites are of S,shows a strong B-cell proliferation effect. However, its IFN-αproduction inducing ability is not so strong. Natural oligonucleic acidconsists of phosphodiester bonds (PO bond) is readily reduced in vivo.

CITATION LIST Patent Literature

-   [Patent Literature 1] JP 2002-513763 A-   [Patent Literature 2] JP 2002-154397 A-   [Patent Literature 3] JP 2002-521489 A-   [Patent Literature 4] JP 2010-504750 A

Non Patent Literature

-   [Non Patent Literature 1] Authur M. Krieg et al. OLIGONUCLROTIDES    13: pp. 491-499 (2003)-   [Non Patent Literature 2] Clin Cancer Res. 2008 Jul. 15;    14(14):4532-42.-   [Non Patent Literature 3] Krieg, A M., J Clin Invest    (2007)117:1184-1194.

SUMMARY OF INVENTION Technical Problem

For example, all of the sequences of the CpG oligonucleotide disclosedin Non Patent Literature 2 are phosphorothioated. Therefore CpGoligonucleotides disclosed in Non Patent Literature 2 have problems thatthey might induce inflammation and toxic reaction. When thephosphorothioate backbone modification is removed from the CpGoligonucleotides disclosed in Non Patent Literature 2, the stability ofthe nucleotide decreases. In addition, there is a problem that NaturalCpG oligonucleotides also readily reduced in vivo. An adjuvant whichsuppresses allergic reaction can be applied to a variety ofapplications. Furthermore, the adjuvant can be used as an antiallergicagent.

One object of the present invention is therefore to provide a stableadjuvant that comprises CpG oligonucleotide having anti-allergic effect.

Other object of the present invention is to provide an anti-allergicagent containing a stable CpG oligonucleotide. In particular, one objectof the present invention is to provide a therapeutic agent for allergicrhinitis or an adjuvant for the therapeutic agent for allergic rhinitis,which comprises a stable CpG oligonucleotide.

Solution to the Problem

This invention is basically based on the following new finding. It ispossible to enhance in-vivo stability of the oligo nucleic acid bycontrolling the molecular conformation of the oligo nucleic acid. Itbecomes possible to provide a stable oligonucleotide in vivo, withoutintroducing the PS bond to all of the sequences. Because not all of thesequences have PS binding modification, the oligonucleotide of thepresent invention has excellent biocompatibility. The oligonucleotidesof the invention also have an anti-allergic action as shown in theworking examples below.

The first aspect of the invention relates to an adjuvant. As describedabove, the adjuvant has excellent biocompatibility and anti-allergiceffect. Because the adjuvant of the present invention has the abovecharacteristics, it has the following features.

That is, the adjuvant comprises an oligonucleotide which contains two tofour CpG motif consisting of 5′-X₁CpGX₂-3′ and has a length of 14 to 32nucleotides, wherein the CpG is non-methylated CpG without modifiedphosphate backbones, wherein the X₁ is A or T, wherein the X₂ is A or T.Phosphorothioated nucleic acids are linked at the 3′ end side of two CpGmotifs of the oligonucleotide. Each of the nucleic acids at 5′ end and3′ end of the oligonucleotide is S type nucleic acids connected byphosphorothioate linkage. The oligonucleotide comprises at least onenucleic acid without phosphorothioate modification. It is preferred thatnucleic acids without modified phosphate backbones present on the partbesides CpG motifs.

In preferred example of the adjuvant of the present invention, X₁ is A,and X₂ is T. Then, the nucleic acid with phosphorothioate bond is afirst S-form T having phosphorothioate bond. That is, in the adjuvant ofthe present invention, a nucleic acid at 3′ end side of at least two CpGmotifs is connected by phosphorothioate linkage. In the preferredexample, T^(SP) (T^(SP) represents T with phosphorothioate bond) ispresent adjacent to the 5′-ACpGT-3,

More preferred example of the adjuvants of the present invention is anadjuvant that has a further second S-form T adjacent to the first S-formT (5′-ACpGTT^(SP)T^(SP)-3). Furthermore, an adjuvant that has a thirdS-form T adjacent to the second S-form T (5′-ACpGTT^(SP)T^(SP)T^(SP)-3)is also preferred.

In the adjuvant of the present invention, it is preferred that thenucleic acid base of 5′ terminal is the S-form nucleic acid base withphosphorothioate bond. Furthermore, in the adjuvant of the presentinvention, it is preferred that the nucleic acid base of 3′ terminal isthe S-form nucleic acid base with phosphorothioate bond.

Specific examples of the adjuvant of the present invention are adjuvantscomprising an oligonucleotide having the nucleotide sequence representedby SEQ ID NO: 4, 5, 9, 10, 20, 22, 23, 29, 30, 31, 39, 53, 54, 55, 59,64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 78, 81 or 84.

The oligonucleotide of this invention is preferred to be anoligonucleotide consisting of any of the following sequences.

TABLE 1 Seq. No. Sequence (5′→3′)  4 T*C*AACGTT*T*C*AACGTT*T*T*G*G  5T*C*AACGTT*T*C*AACGTT*G*G  9 T*C*AACGTT*T*A*AACGTT*T*T 10T*C*AACGTT*T*A*AACGTT*T*A*AACGGG 20 T*C*GACGT*T*GACGT*T*GACGT*T*GACGGG22 T*C*GACGTT*T*A*AACGTT*T*A*GACGTT*T*A*AACGGG 23T*C*GACGTT*AACGTT*AACGTT*AACGGG 29 T*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G 30T*C*GACGTT*T*T*GACGTT*T*T*GACGT*G*G 31T*C*GACGTT*T*T*GACGTT*T*T*GACGTG*G*G 39 T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGGG53 T*C*GACGT*T*GACGT*T*GACG*G*G 51 T*C*GACGT*T*GACGT*T*GACGG*G*G 55T*C*GACGT*T*GACGT*T*GACGT*G 59 T*C*GACGT*T*GACGT*T*GACG*G*G 64C*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G 65 T*C*GACGTT*T*A*GACGTT*T*A*GACG*G*G66 T*C*GACGTT*T*T*GACGTT*T*T*GACG*A*A 67T*C*GACGTT*T*T*GACGTT*T*T*GACG*T*T 68 T*C*AACGTT*T*T*AACGTT*T*T*GACG*G*G69 T*C*GACGTT*T*T*GACGTT*T*T*GGG 70 T*C*GACGTT*T*T*GACGTT*T*T*GACGTTGG71 T*C*GACGTT*GACGTT*G*G*G 74 T*C*G*ACGTT*T*T*GACGTT*T*T*G*ACG*G*G 75T*C*GACGTA*GACGTA*GACG*G*G 76 T*A*GACGAT*T*C*GTCGTC*T*A*GACG*G*G 77T*A*GACGA*C*GTCGT*A*GACG*G*G 78 T*C*G*ACGTTT*T*G*ACGTT*T*T*G*A*C*G*G*G81 T*C*ATCGAT*T*T*ATCGAT*T*T*GACG*G*G 84T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*ATCG*G*G

In above formula * indicates the stereoisomer caused by phosphatebackbone modification by sulfur atoms. In each sequence above, at leastone of the * is S-form stereoisomer.

The adjuvant of the present invention may contain the oligonucleotidesthat have anti-allergic activity and comprise oligonucleotides that oneor two bases are substituted from, inserted to, deleted from or added tooligonucleotides having the nucleotide sequence represented by SEQ IDNO: 4, 5, 9, 10, 20, 22, 23, 29, 30, 31, 39, 53, 54, 55, 59, 64, 65, 66,67, 68, 69, 70, 71, 74, 75, 76, 77, 78, 81 or 84.

As the oligonucleotides of the invention, the oligonucleotiderepresented by the following formula (I) (oligonucleotide having anucleotide sequence represented by SEQ ID NO: 67) is preferred. However,another preferred oligonucleotides of the present invention, isoligonucleotides that have anti-allergic activity and compriseoligonucleotides that one or two bases are substituted from, insertedto, deleted from or added to oligonucleotides having the nucleotidesequence represented by formula (I).T^(SP)C^(SP)GACGTT^(SP)T^(SP)T^(SP)GACGTT^(SP)T^(SP)T^(SP)GACG^(SP)T^(SP)T  [Formula(I)]

In above formula (I), ^(SP) indicates the stereoisomer caused byphosphate backbone modification by sulfur atoms having S-formconformation.

The second aspect of the present invention relates to an antiallergicagent. The term “anti-allergic agent” means a therapeutic agent orprophylactic agent for allergic diseases. Anti-allergic agent of thepresent invention comprise any of the adjuvants described above. Suchanti-allergic agents may comprise known agents as an active ingredientas an anti-allergic agent. In this case, it is possible to enhance theeffect of known allergy agent because the adjuvants also exertanti-allergic action, Further, it may be possible to improve theallergic diseases in patients unable to improve allergic disease byknown allergy agent. Preferred examples of the anti-allergic agent is atherapeutic agent for allergic rhinitis.

The anti-allergic agent of the present invention may be those containingthe oligonucleotide described above as an active ingredient. In thiscase, the anti-allergic agent of the present invention may be used incombination with a therapeutic agent known in allergic diseases. Theanti-allergic agent of the present invention may also comprise onlyoligonucleotides of the present invention as an active ingredient. Inthis case, preferred examples of anti-allergy agent is a therapeuticagent for allergic rhinitis.

Effect of the Invention

According to the present invention, it is possible to provide anadjuvant comprising a stable CpG oligonucleotide having anti-allergiceffect.

According to the present invention, it is possible to provide ananti-allergic agent containing a stable CpG oligonucleotide havinganti-allergic effect. According to the present invention, it is possibleto provide a therapeutic agent for allergic diseases such as allergicrhinitis, or an adjuvant used in the therapeutic agent for allergicdiseases such as allergic rhinitis, which has a stable CpGoligonucleotide having anti-allergic effect.

BRIEF EXPLANATION OF FIGURES

FIG. 1 is a graph that shows an anti-allergic effect of the chiralnucleic acid adjuvant to an OVA induced allergic rhinitis model mouse.

FIG. 2 is a graph that shows an effect of the chiral nucleic acidadjuvant for the density of IgE in the serums of OVA induced allergicrhinitis model mouse.

FIG. 3 is a graph that shows the measured value of OVA specific IgE inthe serums of mouse.

FIG. 4 is a graph that shows the count of leukocytes in BALF,Bronchoalveolar Lavage Fluid.

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes embodiments of the present invention. Thepresent invention is not limited to the embodiments described below andincludes those that a skilled person in the art will easily modify basedon the following embodiments.

The first aspect of the present invention relates to an adjuvant. Asdescribed above, the adjuvant has excellent biocompatibility andanti-allergic effect. Because the adjuvant of the present invention hasthe above characteristics, it has the following features. The adjuvantof the present invention can preferably be used as an adjuvant for theanti-allergic agent. Particularly, the adjuvant of the present inventioncan be preferably used as an adjuvant for the treatment agent forallergic rhinitis. In these cases, the adjuvants of the presentinvention activate or support the function of the active ingredient ofanti-allergic agents. Then, it is possible to provide more effectiveanti-allergic agents.

The adjuvant of the present invention comprises an oligonucleotide whichcontains two to four CpG motif consisting of 5′-X₁CpGX₂-3′ and has alength of 14 to 32 nucleotides. Then, CpG represents non-methylated CpGwithout phosphate backbone modifications. X₁ is either A or T, X₂ iseither A or T. The bases of the 5′ and 3′ terminals of theoligonucleotides are S-form oligonucleic acids having phosphorothioatebond. The oligonucleotides include at least one or more bases that arenot phosphorothioate modification. It is preferred that bases withoutphosphorothioate modification are present in a portion other than CpGmotifs.

“Oligonucleotide” or “oligo” means sugar (e.g. ribose or deoxyribose)binding multiple nucleotides (i.e. Phosphate groups and substitutedorganic bases (either of substituted pyrimidines (e.g. cytosine (C),thymine (T) or uracil (U)) or substituted purine (e.g. adenine (A) orguanine (G))). As used in this specification, the term “oligonucleotide”means both of oligoribonucleotide (ORN) and oligodeoxyribonucleotide(ODN). The term “oligonucleotide” includes also oligonucleoside (i.e.,the oligonucleotide without phosphate) and any other organic basepolymer. Oligonucleotides can be obtained from existing nucleic acidsources (e.g. genome or cDNA), but synthetic one (e.g. produced byoligonucleotide synthesis) is preferred.

CpG represents unmethylated CpG without phosphate backbone modification.C is 2′-deoxycytidine. G is 2′-deoxyguanosine. p is a bond betweennucleoside with phosphodiester.

The oligonucleotide may have phosphate backbone modification on the partbesides the CpG motif consisting of 5′-X₁CpGX₂-3′. On the other hand,there is the problem previously said on phosphate backbone withphosphorothioate backbone modification between all of nucleotides, itmay be preferable that oxygen atoms are replaced by sulfur atoms by morethan 20% less than 95%, it may be more than 30% less than 95%, more than20% less than 90%, more than 40% less than 95%, more than 40% less than90%, more than 40% less than 80%, more than 50% less than 95%, more than50% less than 90%, more than 50% and less than 80%, or more than 60% andless than 95%. more than 20% less than 80%.

The oligonucleotide of the present invention is preferred to have thefollowing, or comprising any sequence, it is preferable that anoligonucleotide has any of the following sequences.

TABLE 2 Seq. No. Sequence (5′→3′)  4 T*C*AACGTT*T*C*AACGTT*T*T*G*G  5T*C*AACGTT*T*C*AACGTT*G*G  9 T*C*AACGTT*T*A*AACGTT*T*T 10T*C*AACGTT*T*A*AACGTT*T*A*AACGGG 20 T*C*GACGT*T*GACGT*T*GACGT*T*GACGGG22 T*C*GACGTT*T*A*AACGTT*T*A*GACGTT*T*A*AACGGG 23T*C*GACGTT*AACGTT*AACGTT*AACGGG 29 T*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G 30T*C*GACGTT*T*T*GACGTT*T*T*GACGT*G*G 31T*C*GACGTT*T*T*GACGTT*T*T*GACGTG*G*G 39 T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGGG53 T*C*GACGT*T*GACGT*T*GACG*G*G 54 T*C*GACGT*T*GACGT*T*GACGG*G*G 55T*C*GACGT*T*GACGT*T*GACGT*G 59 T*CAGACGT*T*GACGT*T*GACG*GAG 64C*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G 65 T*C*GACGTT*T*A*GACGTT*T*A*GACG*G*G66 T*C*GACGTT*T*T*GACGTT*T*T*GACG*A*A 67T*C*GACGTT*T*T*GACGTT*T*T*GACG*T*T 68 T*C*AACGTT*T*T*AACGTT*T*T*GACG*G*G69 T*C*GACGTT*T*T*GACGTT*T*T*GGG 70 T*C*GACGTT*T*T*GACGTT*T*T*GACGTTGG71 T*C*GACGTT*GACGTT*G*G*G 74 T*C*G*ACGTT*T*T*GACGTT*T*T*G*ACG*G*G 75T*C*GACGTA*GACGTA*GACG*G*G 76 T*A*GACGAT*T*C*GTCGTC*T*A*GACG*G*G 77T*A*GACGA*C*GTCGT*A*GACG*G*G 78 T*C*G*ACGTTT*T*G*ACGTT*T*T*G*A*C*G*G*G81 T*C*ATCGAT*T*T*ATCGAT*T*T*GACG*G*G 84T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*ATCG*G*G

In above formula * indicates the stereoisomer by phosphate backbonemodification. CG of the section corresponding to 5′-X₁CpGX₂-3 in aboveformula means unmethylated CpG without phosphate backbone modifications.The examples of phosphate backbone modifications are phosphorothioatebackbone modifications, phosphorodithioate backbone modifications, orphosphoramidate backbone modifications. In these phosphate backbonemodifications, phosphorothioate backbone modifications are preferred.phosphorothioate backbone modifications means that converting one of thetwo nonbridging oxygen atoms bonding to phosphorus atoms comprisingphosphodiester bond of neighbor nucleotides into sulfur atoms. At leastone of the * is S-form stereoisomer. Here, S-form stereoisomer means, asdescribed above, stereoisomer that takes S-form when their atoms orbases introduced instead of oxygen atoms are sulfur atoms.

The oligonucleotide of the present invention is preferred that it is oneof the nucleotides described above, and phosphate backbone modificationswhich exist in at least one of the sites other than CpG motif areoligonucleotides including phosphorothioate. That is, as describedabove, it is preferred that the oligonucleotide has phosphorothioatebackbone modification also in the sites other than CpG. In this case, asdescribed above, it is preferred that the phosphorothioate backbone isS-form stereoisomer. However, in the present invention, oligonucleotideswith at least one unmodified phosphorothioate backbone is preferred thanoligonucleotides that all parts of the sequence are phosphorothioated.

Synthetic Method of Nucleotides

The synthetic method for nucleotides is publicly known. The nucleotidesin present invention can be produced by the publicly known method. Forexample, it can adopt that the methods disclosed in Japanese Patent No.450870 and WO international application No. 2010/064146 pamphlet.

The other examples of the method for synthesizing nucleotide areintroduced in Japanese Patent No. 4942646 and U.S. Pat. No. 5,912,332.The latter, the use of the solid support attachment linker to parallelsynthesis or generic solid support, such as phosphate salt attachingcontrolled pore glass.

Furthermore, nucleotide can be produced by the method e.g. disclosed inU.S. Pat. No. 4,383,534 A. For example, It can be produced byβ-cyanoethyl phosphoroamidate method (S. L. Beaucage, M. H. Caruthers,Tetrahedron Lett. 1981, 22, 1859-62) and nucleoside H-phosphonate method(Per J. Garegg et al., Tetrahedron Lett. 1986, 27, 4051-4; Brian C.Froehler et al., Nucl Acid Res 1986, 14, 5399-407; Per J. Garegg et al.,Tetrahedron Lett. 1988, 27, 4055-8; Barbara L. Gaffney et al.,Tetrahedron Lett., 29, 2619-22). These chemicals can be synthesized by avariety of automated nucleic acid synthesizers available in the market.These nucleic acids are called synthetic nucleic acid. Alternatively, itis possible to generate nucleic acids of the present invention on alarge scale in a plasmid. (Sambrook T. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989)The nucleic acid of this invention can be separated into smaller piecesor administered whole. The nucleic acid is produced from nucleic acidsequence (e.g. genomic sequence and cDNA sequence) with the use of knowntechniques (e.g. techniques using restriction enzymes, exonuclease orendonuclease) The nucleic acid that has been prepared in this way iscalled isolated nucleic acid. An isolated nucleic acid, in general, is anucleic acid which is separated from components which naturally arenormally associated. For example, the isolated nucleic acid is a nucleicacid that is separated from the cells, nucleus, mitochondria andchromatin. The combination motif nucleic acid of the present inventionincludes both synthesized combination motif nucleic acids and isolatedcombination motif nucleic acids.

The combination motif oligonucleotides, if necessary, have a relativelyresistant to degradation (e.g., are stabilized) are preferred in the useof in vivo. A “stabilized nucleic acid molecule” means a nucleic acidmolecule that is relatively resistant for in vivo degradation (e.g.,exonuclease or endonuclease). The nucleic acid stabilization is achievedthrough the phosphate backbone modification. The stabilized nucleic acidthat is preferred in the present invention has a modified backbone. Thismodification of the nucleic acid backbone provides increasing theactivity of the combination motif oligonucleotide when administered invivo. In some cases, the combination motif oligonucleotides withphosphorothioate bond provide maximum activity and protect the nucleicacid from degradation by intracellular exonucleases and cellularendonucleases. Other modified nucleic acids, modified phosphodiesternucleic acids, combinations of phosphodiester nucleic acids andphosphorothioate nucleic acids (i.e. chimeric), methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof mentioned are.

The modified backbones (e.g., phosphorothioates) can be synthesized byusing automated techniques employing either phosphoramidate chemistry orH-phosphonate chemistry. Aryl-phosphonate and alkyl-phosphonates can begenerated, for example, as described in U.S. Pat. No. 4,469,863. Andalkylphosphotriester (charged oxygen is alkylated as described in U.S.Pat. No. 5,023,243 and EP patent No. 092,574) can be produced usingcommercially available reagents by automated solid-phase synthesis.Methods for making modifications and substitutions of other DNA backbonehave been described. (e.g., Uhlmann E and Peyman A, Chem. Rev. 1990, 90,544; Goodchild J., Bioconjugate Chem. 1990, 1, 165).

The oligonucleotides obtained by synthesis may be purified by knownmethods, e.g., purified, deprotected, desalted and dialyzed by reversedphase HPLC. In this way, the oligonucleotides of the present inventioncan be isolated and purified.

This invention provides composition with one of the aboveoligonucleotides. This composition is a medicine composition. Thecomposition contains an effective amount of any of the oligonucleotidesdescribed above and it may contain appropriate known carrier. Thecarrier may be a solvent such as water or alcohol. The carrier can beoptional excipients, diluents, fillers, salts, buffers, stabilizers,solubilizers, lipids or other substance which is well known for medicinecompositions in the art.

This invention also provides a vaccine adjuvant with oligonucleotidedescribed above. The vaccine adjuvant, if necessary, may contain apharmaceutically acceptable carrier. U.S. Pat. No. 4,126,252 disclosesvaccine adjuvant with oligonucleotide. The vaccine adjuvant with theoligonucleotide of this invention can include the disclosed elements inthis publication properly.

The examples of allergic disease related to anti-allergic effect of thisinvention are systemic inflammatory response syndrome (SIRS),anaphylaxis or anaphylactoid reaction, allergic vasculitis, hepatitis,nephritis, rhinitis, arthritis, inflammatory eye diseases (e.g.,conjunctivitis, etc.), inflammatory bowel disease (e.g., eosinophilicgastrointestinal disease, etc.), brain and cardiovascular systemdiseases, skin disease (e.g., dermatitis (e.g., atopic dermatitis,contact dermatitis, eczema, urticaria, pruritus, etc.), etc.),autoimmune diseases (such as rheumatoid arthritis, systemic lupuserythematosus, glomerulonephritis, Sjogren's syndrome, etc.),transplanted organ rejection.

Allergic diseases can be classified into type I˜type IV.

The examples of Type I allergy are an allergic rhinitis, hay fever,bronchial asthma, urticaria, atopic dermatitis, allergic conjunctivitis,food allergy, and anaphylaxis.

The examples of Type II allergy are autoimmune hemolytic anemia,thrombocytopenia, granulocytopenia, hemolytic neonatal jaundice,pernicious anemia, rheumatic fever, myasthenia gravis, Hashimoto'sdisease, and an alopecia areata.

The examples of Type III allergy are serum sickness, hypersensitivitypneumonitis, lupus nephritis (chronic glomerulonephritis), systemiclupus erythematosus, acute glomerulonephritis, rheumatoid arthritis,rheumatic pneumonia, multiple arteritis, allergic vasculitis, and somein Sjogren's syndrome.

The examples of Type IV allergy are contact dermatitis, Hashimoto'sdisease, Behcet's disease, rejection after organ transplantation,graft-versus-host disease (GVHD), and Guillain-Barre syndrome.

Anti-allergic effects of the present invention is directed to all ofallergy of type I˜type IV. In particular, the present invention can besuppressed type I allergy.

As shown in embodiments below, the oligonucleotides of present inventionand the adjuvants containing them particularly have therapeutic effectsin allergic rhinitis. Therefore, the oligonucleotides and the adjuvantsare effective as allergic rhinitis therapeutic agents and adjuvant forthe treatment agent for allergic rhinitis, respectively.

This oligonucleotide is effective to induce systemic immune responseand/or mucosal immune response. The combination motif oligonucleotide ofthis invention can be delivered to a subject exposed to antigens forinducing enhancement of the immune response to antigens. Therefore, forexample, the combination motif nucleotide is useful for vaccineadjuvant. Examples of the main agent functioning as adjuvant are avariety of vaccines. The adjuvant can increase the efficiency of antigento be incorporated into the immune cells. The adjuvant is preferred tobe able to be improved or enhanced or assist the original action withthe active ingredient of the main agent.

The examples for the vacctine are vacctine for virus and vacctine for Bhepatitis, A hepatitis, Japanese encephalitis, pediatric pneumococcal,diphtheria, cough hundred days, tetanus, measles, rubella, mumps,chicken pox and tuberculosis (BCG vaccine). Examples of the vaccine forvirus are influenza vaccine, polio vaccine, human papillomavirusvaccine, rotavirus vaccine, vaccine for non-flops, polio vaccines andAIDS vaccine. The oligonucleotide of this invention functions asadjuvant by a very small amount. Therefore, the oligonucleotide of thisinvention have low cytotoxic compared with the conventional adjuvant andthere are very few side effects. This makes the vaccine which isadministrated in many targets very useful.

This oligonucleotide strongly induces type 1 helper T cells (Th1). As aresult of this, the oligonucleotide attenuate the action of T-helpercell type 2 (Th2). In other words, it is expected that theoligonucleotide can improve the pathology of Type I allergy caused bythe activation of Th2. The antiallergic agents in the prior art can nottreat a condition and only be able to improve allergy symptoms. Thisoligonucleotides has the effect of restoring the allergic condition,which caused by the collapse of the balance of Th1 and Th2, to thenormal state. The examples of Type I allergy are Allergic rhinitis, hayfever, bronchial asthma, urticaria, atopic dermatitis, allergicconjunctivitis, food allergy and anaphylaxis. In addition,oligonucleotides of the present invention can act in a very small amountas an anti-allergy adjuvant. Therefore, the oligonucleotides of thepresent invention have lower cell toxicity compared with theconventional adjuvant, and fear of side effects very small. This featureis extremely useful in allergy treatment.

This oligonucleotide can be administrated with a known anti-allergytreatment. The term “non-nucleic acid adjuvant” means any molecule orcompound except the oligonucleotides described herein, which canstimulate the humoral immune response and/or cellular immune response.The examples of anti-allergic agent are antihistamines, steroids,anti-leukotriene agents, and the like.

The oligonucleotide of this invention may be formulated as apharmaceutical composition in a pharmaceutically acceptable carrier.This oligonucleotide may be administrated to a subject directly or witha nucleic acid delivery complex. The nucleic acid delivery complex meansa nucleic acid which is associated (e.g., ionic bond or covalent bond,or encapsulated in the way) with a targeting way (e.g., molecules whichgenerate high affinity bond to target cells (e.g., surface of B cell)and/or increase in cellular uptake by target cells.). The examples ofthe nucleic acid delivery complex are nucleic acid associated withsterols such as cholesterol, lipids (e.g., cationic lipids, virosomes orliposomes) or target cell specific bonding factors (egg, ligandsrecognized by target cell specific receptor). Preferred complex can beenough stable in vivo to prevent from significant de-coupling before theinternalization by the target cell. But the complex can be cleavageunder appropriate conditions in the cells so that the nucleic acid isreleased in a functional form.

This oligonucleotide and/or the antigen and/or other therapeutic agentscan be administrated separately (e.g. in saline or buffer solution), andmay also be administered using any known delivery vehicles.

Dose of the compounds described herein for systemic administration,mucosal delivery or topical delivery is typically in the range of 0.1μg/dose to 10 mg/dose. The doses depend on whether it is administereddaily, weekly, or monthly, and in any other time. More typically, dosesfor systemic administration, mucosal delivery or topical delivery are inthe range of 10 μg/dose to 5 mg/dose. The most typically, it is 100μg/dose to 1 mg/dose, and the administrations of 2-4 times are performedapart for a few days or a few weeks. More typically, dose is in therange of 1 μg/dose to 10 mg/dose, most typically, in the range of 10 μgdose to 1 mg/dose. Then, the administrations are performed daily orweekly. The dose of the compounds described herein for parenteraldelivery in order to induce an anti-allergic effect, is typically 5 to10,000-fold more than effective mucosal dose for vaccine adjuvant orimmune stimulating applied. More typically, it is 10 to 1,000-foldgreater, and most typically 20 to 100 times greater. In case of that theoligonucleotide is administered in combination with other therapeuticagents or administered using specialized delivery vehicles, the dose ofthe compounds described herein for parenteral delivery is typically inthe range of about 0.1 μg/dose to 10 mg/dose. The doses depend onwhether it is administered daily, weekly, or monthly, and in any othertime. More typically parenteral doses for these purposes are in therange of about 10 μg/dose to 5 mg/dose. The most typically, it is about100 μg/dose to 1 mg/dose, and the administrations of 2-4 times areperformed apart for a few days or a few weeks. However, in someembodiments, parenteral doses for these purposes may be used in the 5 to10,000-fold greater range than the typical doses described above.

In the case of respiratory allergic diseases such as rhinitis andbronchial asthma, a dosage of the oligonucleotides of the presentinvention is preferably 1 μg or more and 10 mg or less. The dosage maybe 10 μg or more and 1 mg or less. Further, the dosage may be 50 μg ormore and 0.5 mg or less.

In the present specification, the term “effective amount” means therequired or sufficient amount to achieve the desired biological effect.For example, an effective amount of a chiral nucleic acid for treatingan allergic disease means the amount required to treat the disease.Combined with the teachings provided herein, by selecting the variousactive compounds and weighing factors (For example, potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration), the effective prevention regimenand the effective therapeutic regimen, which are very effective to treata particular subject without causing substantial toxicity, can be plan.The effective amount for any particular application may vary dependingon factors, such as disease or condition being treated, the particularoligonucleotide being administered, antigen, subject size, and theseverity of the disease and conditions. A skilled in the art can beempirically determined the effective amount of a particularoligonucleotide and/or antigen and/or other therapeutic agents withoutthe need for undue experiments.

A therapeutically effective amount for any of the compounds describedherein can first be determined based on the knowledge obtained in animalexperiments. An effective dose for the treatment also can be determinedbased on the data about the CpG oligonucleotide which has been tested inhuman (human clinical trials has been started) and the data when themucosal or local administration of known compounds having similarpharmacological activities [For example, other mucosal adjuvants (forexample, LT and other antigens for vaccination)]. For parenteraladministration, it is necessary to use higher dose. The applied dose canbe adjusted based on the relative bioavailability and potency of thecompounds administered. Adjusting the dose to achieve the maximalefficacy using the methods and other methods is well known in the art.In addition, a skilled person can easily adjust the dose.

When administered, formulation of the present invention is dissolved ina pharmaceutically demand solutions. The solution conventionally mayinclude salts of pharmaceutically acceptable concentrations, bufferingagents, preservatives, compatible carriers, adjuvants, and optionallyother therapeutic ingredients.

For use in therapy, the oligonucleotide of the effective amount may beadministered to a subject using any manner for delivering the nucleicacids to desired surface (for example, a mucosal surface and asystemically surface). Administering the pharmaceutical compositions ofthis invention may be accomplished by any means known to those skilledin the art. Preferred routes of administration are oral route,parenteral route, intramuscular route, intranasal route, theintratracheal route, inhalation routes, ocular route, sublingual,vaginal routes, rectal route, and the like, but not limited those listedherein.

For oral administration, the compounds (i.e., oligonucleotides,antigens, and other therapeutic agents) can be easily prepared bycombining the active compound with known pharmaceutically acceptablecarriers in the art. Such carriers enable the compounds of the presentinvention to be formulated as tablets to be taken orally by a subject tobe targeted, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like. The pharmaceutical preparations for oraladministration may be obtained as solid excipient by adding suitableauxiliaries if necessary, subsequently grounding the resulting mixtureand forming the tablet cores or the dragee cores by processing themixture of granules. In particular, suitable excipients are fillers [forexample, sugar (lactose, sucrose, mannitol and sorbitol); cellulosepreparations (for example, corn starch, wheat starch, Rice starch,potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose) and/orpolyvinylpyrrolidone (PVP)]. If necessary, the disintegrating agents[for example, cross-linked polyvinyl pyrrolidone, agar, alginic acid ora salt thereof (for example, sodium alginate)] may be added. Ifnecessary, the oral formulations may also be administered in saline orbuffer solution to neutralize the acidic internal state. In addition,the oral formulations may be administered without any carriers.

The dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions can be used. If necessary, the concentratedsugar solutions may contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol and/or titanium dioxide, lacquersolutions, suitable organic solvents or solvent mixtures. In order toidentify or characterize different combinations of active compounddoses, dyestuffs or pigments may be added to the tablets or the drageecoatings.

Examples of pharmaceutical preparations which can be administered orallyare a fabricated capsule of gelatin, and a soft sealed capsule made ofgelatin and a plasticizer (for example, glycerol or sorbitol). Thecapsule may contain the active ingredient, if necessary, mixed withfillers (for example, lactose), binders (for example, starch) and/orlubricants (for example, talc or magnesium stearate) and stabilizers. Inthe soft capsule, the active compounds may be dissolved or suspended insuitable liquids (for example, fatty oils, liquid paraffin, or liquidpolyethylene glycol). In addition, the stabilizer may be added.Microspheres formulated for oral administration may also be used. Suchmicrospheres have been well known in the art. All formulations for oraladministration may be used in appropriate dosage.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For inhalation administration, the compounds of the present inventionmay be administrated by aerosol spray from pressurized packs or anebulizer using a suitable propellant (for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas) as witha conventional usage. When using a pressurized aerosol, the dosage unitcan be determined by providing a valve to deliver a metered amount. Foruse in an inhaler or insufflator, such gelatin capsules and cartridges,which contain a powder mixture of the compound and a suitable powderbase, may be provided.

If the compound should be delivered systemically, the compound can beprovided in a form that can be administered parenterally by injection(for example, bolus injection or continuous infusion). The formulationsfor injection may be provided in unit dosage form (for example, anampoule or multi-dose containers) with preservative agent. The compoundsmay take such forms as solutions, emulsions or suspension in oily oraqueous vehicles. In addition, they may contain the formulations (forexample, suspending agents, stabilizing agents and/or dispersingagents).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds which are water soluble. Inaddition, suspensions of the active compounds may be provided asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils (for example, sesame oil), synthetic fattyacid esters for example, ethyl oleate or triglycerides), or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension (for example, sodium carboxymethylcellulose, sorbitol, or dextran). In order to prepare highlyconcentrated solutions, the suspension may also include agents thatincrease the solubility of the appropriate stabilizers or compoundsthereof as necessary.

Alternatively, the active compounds may be in powder form which can beconfigured prior to use with a suitable vehicle (for example, sterilepyrogen-free water).

The compounds may be provided in the form for rectal or vaginaladministration (for example, suppositories or retention enemas which maycontain conventional suppository bases such as cocoa butter or otherglycerides).

In addition to the above, the compounds may also be provided as a depotpreparation. Such long acting formulations may be provided by using asuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil), by using an ion exchange resin or by using poorlysoluble derivatives (such as salts poorly soluble).

The pharmaceutical compositions may also include carriers or excipientswhich is a suitable solid or gel phase. Examples of such carriers orexcipients include calcium carbonate, calcium phosphate, various sugars,starches, cellulose derivatives, gelatin, and polymers (for example,polyethylene glycol), but not limited thereto.

Suitable liquid pharmaceutical preparation form or solid pharmaceuticalpreparation forms are micro-encapsulated, chelated, coated onmicroscopic gold particles, included in liposomes, contained in theaerosol to be sprayed, included in the pellet for implantation into theskin, dried form in sharp on the object for scratching the skin, aqueoussolution for inhalation or saline solution. In addition, thepharmaceutical compositions includes granules, powders, tablets, coatedtablets, (micro) capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations, which can release the activecompound a long period of time. As described above, the formulationscontain the excipients, the additives and/or the adjuvants (for example,disintegrants, binders, coating agents, sweetening agents, lubricants,flavoring agents, sweeteners, or solubilized agents) conventionally. Thepharmaceutical compositions are suitable for use in a variety of drugdelivery systems. Brief review of methods for drug delivery is mentionedin Langer (1990) Science 249: 1527-33 (which is incorporated herein byreference).

The oligonucleotide and that containing other therapeutic agent and/orantigen as necessary may be administered without being any processed, ormay be administered in the form of a pharmaceutically acceptable salt.When administered in the form of a pharmaceutically acceptable salt, thesalt should be pharmaceutically acceptable. However, thepharmaceutically acceptable salt may be used to prepare thepharmaceutically acceptable salts. Examples of such salts are thefollowings, but not limited thereto: HCl, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylicacid, p-toluene sulfonic acid, salt tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid,naphthalene-2-sulfonic acid and benzene sulfonic acid. In addition, suchsalts may be prepared as alkali metal salts or alkaline earth metalsalts (for example, sodium salts of carboxylic acid, potassium salt orcalcium salt).

Examples of the suitable buffering agents are followings: acetic acidand its salt (1-2% w/v); citric acid and its salt (1-3% w/v); boric acidand its salt (0.5-2.5% w/v); and phosphoric acid and its salt (0.8-2%w/v). Examples of the suitable preservatives are followings:benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v);parabens (0.01-0.25% w/v), and thimerosal (0.004-0.02% w/v).

The pharmaceutical compositions of the present invention may contain aneffective amount of the oligonucleotide, the antigen and/or other agentsin a pharmaceutically acceptable carrier as necessary. The term“pharmaceutically acceptable carrier” means one or more compatiblefiller, diluent, or encapsulating agent which is solid or liquid and issuitable for administration to humans or other vertebrates. The term“carrier” means a natural or synthetic, organic or inorganic componentwhich is added to in order to facilitate the application of the activeingredient. Components of the pharmaceutical compositions can be mixedwith the compounds of this invention and each component in a manner thatthe components do not interact with each other.

For the treatment of individual subjects, different capacities of thepharmaceutical compositions of the present invention are required basedon activity of the compound, manner of administration, purpose of theimmunization (i.e., prophylactic immunization or immunotherapy), thenature and severity of the disorder, the age of the patient and weightof the patient. Administration of a desired dose may be performed byadministering an amount corresponding to dosage units at a time or byadministering a smaller amount multiple times.

Examples of other delivery systems include time-release system, delayedrelease system, or sustained release system. Such systems may avoidrepeated administrations of the compound, and may increase theconvenience to the subject and the physician. Many types of releasedelivery systems are available, and are known to those skilled in theart. Examples of the release delivery systems include a polymer-basedsystem (for example, poly (lactide-glycolide), copoly oxalate,polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides). For example, microcapsules of the polymercontaining the pharmaceutical compositions are described in U.S. Pat.No. 5,075,109. The delivery systems also include non-polymeric system.Examples of the non-polymeric system are followings: lipids (sterols(for example, cholesterol, cholesterol ester), and fatty acids ornatural fats (for example, monoglycerides, diglycerides, andtriglycerides) and the like); hydrogel release systems; silastic system;peptide-based systems; wax coating; compressed tablets usingconventional binders and excipients; partial fused to the implant. Inparticular, the system includes the followings, but not limited thereto:(a) an erosion-based system which the agent of the present invention iscontained in a form located in the matrix (U.S. Pat. Nos. 4,452,775,4,675,189 and 5,736,152); (b) a diffusion system which the activeingredient penetrate at a controlled rate from the polymer (U.S. Pat.Nos. 3,854,480, 5,133,974 and 5,407,686). In addition, pump-basedhardware delivery systems can be used. Some of them are adapted forimplantation.

The invention is further illustrated by the following examples. Thefollowing examples are should not be construed as further limiting.Throughout this specification, all of the contents of the citeddocuments are incorporated herein.

Working Example 1

Synthesis of Chiral CpG Oligonucleic Acid

CpG Oligonucleic Acid (Mixture)

The oligonucleic acid (mixture) which had been synthesized usingphosphoramidite method and purified using HPLC were purchased fromGeneDesign, Inc.

Synthesis of the CpG oligonucleotide of which the three-dimensionalstructure is modified.

Extension of nucleic acid chain was performed by repeating the followingsteps (i)-(iv).

-   (i) 3% DCA (dichloroacetic acid)/CH₂Cl₂ (15 sec),-   (ii) Condensation reaction [A mixture of 0.1 M monomer solution in    MeCN (See below) and 1 M PhIMT (Trifluoromethanesulfonic acid    N-phenylimidazolium) solution in MeCN in ratio 1:1, 5 min],-   (iii) Capping reaction [A mixture of 0.5 M CF₃Colm in THF and 1 M    DMAN (1,8-bis(dimethylamino)naphthalene)) in THF in ratio 1:1, 30    sec],-   (iv) Sulfurization reaction (0.1 M DDTT in MeCN, 90 sec) or    oxidization reaction (0.02 M I₂ in H₂O-Pyridine-THF solution, 15    sec).

After the chain elongation of nucleic acid, a solid phase carrier wascollected in 1.5 ml microtube. The solid phase carrier was treated withhigh concentrated aqueous ammonia (1.2 ml, 55 degrees, 48 hours). Thesolid phase carrier was removed by filtration. A filtrate was dried inreduced pressure, and dissolved in water (1.0 ml). Then, the oligomerwas isolated and purified by using a reversed-phase HPLC.

A procedure for adjusting 0.1 M monomer solution in MeCN (in case ofRp-Th).

Thymidylic acid H-phosphonate monoester (25 μmol) was azeotropic-driedwith dehydrated pyridine and dehydrated toluene. It was dissolved inMeCN-CMP (N-Cyanomethylpiperidine) solution (9:1, v/v; 250 μL).Subsequently, Ph₃PCl₂ (62.5 μmol) was added, and the solution wasstirred for 10 min. Then, AA-L (30 μmol; AA-D was used when Sp form wasselected.) was added, and the solution was stirred for 10 min. In thisway, the monomer solution was obtained.

In the description above, DDTT, AA-L and AA-D mean the abbreviateddesignation of the following compounds respectively. The obtainedoligonucleic acids are shown in Table 1.

TABLE 3 Table 1 Oligo nucleic acids obtained in Working Example 1 SEQ.NO. Sequence  1 T*C*GTCGTT*T*T*GTCGTT*T*T*GTCGGG  2G*G*GTCGTT*T*T*GTCGTT*T*T*GTCGGG  3 T*C*AACGTT*T*C*AACGTT*T*T  4T*C*AACGTT*T*C*AACGTT*T*T*GG  5 T*C*AACGTT*T*C*AACGTT*G*G  6T*C*AACGTT*T*C*AACGTT*G*G*G*G  7 T*C*AACGTT*T*T*AACGTT*T*T*AACGGG  8T*C*AACGTT*T*A*ACGTT*T*T  9 T*C*AACGT*TAACGTT*T*T 10T*C*AACGTT*T*A*AACGTT*T*A*AACGGG 11 T*C*AACGTTAACGTTAACGGG 12T*C*GACGTT*T*T*GACGTT*T*T*GACGGG 13 TsCsGACGTTsTsTsGACGTTsTsTsGACGGG 14TrCrGACGTTrTrTrGACGTTrTrTrGACGGG 15 G*G*GACGT*T*T*TGACGT*T*T*TGACGGGGG16 T*C*GACGT*T*T*TGACGT*T*T*TGACGT*T*T*TGACGGG 17T*C*GACGT*T*GACGT*T*GACGGG 18 TsCsGACGTsTsGACGTsTsGACGGG 19TrCrGACGTrTrGATrTrGACGGG 20 T*C*GACGT*T*GACGT*T*GACGT*T*GACGGG 21T*C*GAGGTT*T*A*AACGTT*T*A*AACGTT*T*A*AACGGG 22T*C*GACGTT*T*A*AACGTT*T*A*GACGTT*T*A*AACGGG 23T*C*GACGTTAACGTTAACGTTAACGGG 24 GGGACGTT*T*A*AACGTCTAGACGGG 25T*C*GACGT*ACGT*ACGT*ACGGG 26 T*C*GACGTT*T*T*GACGTT*T*T*G*A*C*G*G*G 27TsCsGACGTTsTsTsGACGTTsTsTsGsAsCsGsGsG 28TrCrGACGTTrTrTrGACGTTrTrTrGrArCrGrGrG 29T*C*GACGTT*T*T*GACGTT*T*T*GACGG*G*G

TABLE 4 30 T*C*GACGTT*T*T*GACGTT*T*T*GACGT*G*G 31T*C*GACGTT*T*T*GACGTT*T*T*GACGTG*G*G 32T*C*GACGTT*T*T*GACGTT*T*T*G*A*C*G*G*G* 33T*C*GACGTT*T*T*GACGTT*T*T*GACGG*G*G*G*G 34TsCsGACGTTsTsTsGACGTTsTsTsGACGGsGsGsGsG 35TrCrGACGTTrTrTrGACGTTrTrTrGACGGrGrGrGrG 36T*c*GACGTT*T*T*GACGTT*T*T*GACG*G*G*G*G 37 T*C*GACGT*T*GACGT*T*GACGTG*G*G38 G*G*T*G*C*ATCGAT*G*C*A*G*G*G*G*G*G 39T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGGG 40 G*G*T*G*C*GACGAT*G*C*A*G*G*G*G*G 41G*G*G*G*GACGATCGTCGGG*G*G*G 42 G*G*GACGATATCGTCG*G*G*G*G*G 43G*G*GACGACGTCGTCG*G*G*G*G*G 41 GsGsGACGACGTCGTCGsGsGsGsGsG 45GrGrGACGACGTCGTCGrGrGrGrGrG 46 G*G*GGGACGATCGTCG*G*G*G*G*G 47G*G*GACGCGCGTCG*G*G*G*G*G*G*G 48 G*G*G*G*TCGTTCG*G*G*G

TABLE 5 Seq. No Sequence 49 T*C*ATCGAT*T*T*ATCGAT*T*T*A*A*C*G*G*G 50T*C*GACGTTTTGACGTT*T*T*G*A*C*G*G*G 51 T*C*GACGTTTTGACGTTTT*G*A*C*G*G*G52 T*C*GACGT*T*GACGT*T*GACGG*G 53 T*C*GACGT*T*GACGT*T*GACG*G*G 54T*C*GACGT*T*GACGT*T*GACGG*G*G 55 T*C*GACGT*T*GACGT*T*GACT*G 56T*C*GACGT*T*GACGT*T*G*A*C*G*G*G 57 T*C*GACGTTGAGGT*T*G*A*C*G*G*G 58T*C*ATCGATATCGA*T*G*A*C*G*G*G 59 T*C*GACGT*T*GACGT*T*GACG*G*G*6 60T*C*GACGTT*T*T*GACGTT*T*T*G*G*G*G*G 61T*C*GACGTT*T*T*GACGTT*T*T*G*A*G*G*G*G 62T*C*GACGTT*T*T*GACGTT*T*T*G*T*G*G*G*G 63 T*C*G*ACGTT*G*ACGTT*G*A*C*G*G*G64 C*C*GACGTT*T*T*GACGTT*T*T*GACG*G*G 65T*C*GACGTT*T*A*GACGTT*T*A*GACG*G*G 66 T*C*GACGTT*T*T*GACGTT*T*T*GACG*A*A67 T*C*GACGTT*T*T*GACGTT*T*T*GACG*T*T 68T*C*AACGTT*T*T*AACGTT*T*T*GACG*G*G 69 T*C*GACGTT*T*T*GACGTT*T*T*GGG 70T*C*GACGTT*T*T*GACGTT*T*T*GACGTTGG 71 T*C*GACGTT*GACGTT*G*G*G 72T*C*GACGTT*T*T*G*ACGTT*T*T*G*ACG*G*G 73 T*C*G*A*CGTT*T*T*G*ACGTTTTGACGGG74 T*C*G*ACGTT*T*T*G*ACGTT*T*T*G*ACG*G*G 75 T*C*GACGTA*GACGTA*GACG*G*G76 T*A*GACGAT*T*C*GTCGTC*T*A*GACG*G*G 77 T*A*GACGA*C*GTCGT*A*GACG*G*G 78T*C*G*ACGTTT*T*G*ACGTT*T*T*G*A*C*G*G*G 79T*C*G*ACGTT*T*T*T*A*ACGAC*T*T*G*A*C*G*G*G

TABLE 6  80 T*C*G*ACGTT T*T*AACGAC*T*T*G*A*C*G*G*G 81 T*C*ATCGAT*T*T*ATCGAT*T*T*GACG*G*G 82 T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGA*T*G*G*G 83 T*C*ATCGAT*T*T*ATCGAT*T*T*AT*C*G*G*G 84 T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*ATCG*G*G 85 T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*T*T*A*T*C*G*G*G  86T*C*ATCGAT*T*T*ATCGAT*T*T*ATCGAT*A*T*C*G*G*G 87 T*T*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*G*G 88 T*C*ATCGATATCGAT*T*T*G*A*C*G*G*G 89 TCATCGAT*T*T*ATCGAT*T*T*A*T*C*G*G*G 90 T*C*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*A*T 91 T*C*GACGT*T*GACGT*T*GACGT*T*G*G*G 92 T*C*G*ACGT*T*G*ACGT*T*G*A*C*G*G*G 93 T*C*A*TCGAT*T*T*A*TCGAT*T*T*G*A*C*G*G*G 94 T*C*A*TCGAT*A*TCGAT*G*ACGT*T*T*G*G*G 95 T*C*GAGGTTTGACGTTT*G*A*C*G*G*G 96 T*C*ATCGAT*T*T*ATCGAT*T*T*A*T*C*G*G*G 97 G*G*GACGATATCGTCG*G*G*G*G*G  98 G*G*GACGAC*G*TCGTCG*G*G*G*G*G 99 G*G*GACGACGTCGTCG*G*G*G*G 100 T*C*GACGACGTCGTCG*G*G*G*G*G

TABLE 7 101 T*C*GACGACGTCGTCT*T*T*G*G*6 102 T*A*GACGACGTCGTCT*T*T*G*G*G103 T*T*GACGACGTCGTCA*A*A*G*G*6 104 T*C*GACGTAGACGTCT*T*T*G*G*6 105T*C*GACGTAGACGTTT*A*G*G*G*G 106 T*C*ATCGATATCGATT*T*T*G*G*G 107T*T*ATCGATATCGATA*A*A*G*G*G 108 T*C*GACGTAGACGATCGA*T*G*G*G 109T*C*GACGAC*T*T*GACGAC*T*T*G*A*C*G*G*G 110T*C*GACGAC*T*T*GTCGTC*T*T*G*A*C*G*G*G 111T*T*ATCGATATCGATA*T*C*G*A*T*G*G*G 112 T*T*ATCGATATCGATT*T*A*A*A*G*G*G113 T*C*ATCGAT*T*T*ATCGAT*T*T*G*A*C*G*T*T 114T*C*ATCGA*T*ATCGA*T*G*A*C*G*G*G*G 115 T*C*ATCGAT*ATCGA*T*G*G*G 116T*C*GTCGTTGTCGT*T*G*A*C*G*G*G 117T*C*G*TCGTT*T*T*G*TCGTT*T*T*G*A*C*G*G*G 118T*C*GTCGTTGTCGTTG*A*C*G*A*C*G*G*G 119T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C* G*T*T

In above tables, oligonucleotides of SEQ ID NO: 119 is a known compound(Compound 119).

In Table 1, * indicates the position that the S-form phosphorothioatebackbone modification or the R-form phosphorothioate modification wereinduced into the backbone at random. In Table 1, “s” indicates theS-form phosphorothioate modification. In Table 1, “r” indicates theR-form phosphorothioate modification.

Working Example 2

Induction of Production of IFN-α in Simian Peripheral Blood MononuclearCells (PBMC)

The blood derived from Macaca fascicularis which has tested negative forB virus, diluted to 3 times with Hanks' Balanced Salt Solution. Then,the sample was layered on Ficoll-Paque PLUS centrifugation medium andcentrifuged (2,600 rpm, 30 min). A fraction containing the peripheralblood mononuclear cells (PBMC) was obtained. After the PBMC was washedwith RPMI medium (1% penicillin and streptomycin), the PBMC wassuspended in RPMI medium (10% FBS, 1% penicillin and streptomycin) at acell density of 3×10⁶ cells/ml. The cells were cultured with variousoligonucleic acids (mixture containing oligo DNA and DOTAP in a ratio of1:3.2) in 96-well round-bottom plate for 17-24 hours using 5% CO₂incubator. After the cultivation, cell culture supernatant was obtainedby centrifuging (500 rpm, 5 min) the culture medium. Then, theconcentration of IFN-α in the cell culture supernatant was measuredusing ELISA kit (PBL Assay Science).

The results are shown in Table 2 and 3. Table 2 indicates the IFN-αproduction induced by the oligonucleic acids of the present invention insimian peripheral blood mononuclear cells (PBMC). This results wereobtained by using the oligonucleotides of SEQ ID: 1-48.

TABLE 2 Inducing effect by the ologonucleotide of the present inventionin IFN-α production in simian peripheral blood mononuclear cells (PBMC)IFN-α (pg/mL) Seq. No. Average ±SE 4 2151 1259.46 5 388 144.35 9 28451127.16 10 256 173.70 20 2457 507.22 22 2492 985.99 23 3082 1379.50 291359 250.85 39 1778 478.20 31 1934 600.05 39 1580 2131.02

Table 3 indicates the IFN-α production induced by the oligonucleic acidsof the present invention in simian peripheral blood mononuclear cells(PBMC). This results were obtained by using the oligonucleotides of SEQID: 49-118. The results in Table 3 were shown using relative values withthe value in the case of using the oligonucleotide having the sequence(tcgtcgllllgtcgttttgtcgtt) of SEQ ID: 119 (conventional nucleotide:Compound 119).

TABLE 3 Inducing Effect of IFN-α Number of Seq. NO. Ratio ±SE samples 53361 60 5 54 225 101 3 55 124 39 4 59 139 23 4 64 84 36 4 65 246 20 4 66188 56 5 67 244 93 5 68 220 97 5 69 98 44 5 70 242 80 4 71 238 48 3 74247 120 5 75 188 54 4 76 260 49 3 77 86 17 4 78 98 6 3 81 389 163 3 84334 40 3

Comparative Example 1

Using the nucleotide having the sequence of SEQ ID: 119 (conventionalnucleotide: Compound 119), the concentration of IFN-α was measured inthe same manner as in above example 2. The results are shown in Table 4.

TABLE 4 The concentration of IFN-α IFN-α (ng/mL) ±SE 52.2 6.2

Working Example 3

Cytokine production profile in simian peripheral blood mononuclear cells(PBMC) The blood derived from Macaca fascicularis which has testednegative for B virus, diluted to 2 times with Hanks' Balanced SaltSolution. Then, the sample was layered on Ficoll-Paque PLUScentrifugation medium and centrifuged (2,600 rpm, 30 min). A fractioncontaining the peripheral blood mononuclear cells (PBMC) was obtained.After the PBMC was washed with RPMI medium (100 IU/ml penicillin and 100μg/ml streptomycin), the PBMC was suspended in RPMI medium (10% FBS, 100IU/ml penicillin and 100 μg/ml streptomycin) at a cell density of 3×10⁶cells/ml. The cells were cultured with various compounds (mixturecontaining CpG oligonucleotide and DOTAP in a ratio of 1:3.2) in 96-wellround-bottom plate for 16-20 hours using 5% CO₂ incubator. After thecultivation, cell culture supernatant was obtained by centrifuging (500rpm, 5 min) the culture medium. Then, in order to obtain the cytokineprofile in the cell culture supernatant, 7 kinds of cytokine, i.e.,IFN-γ, IL-4, IL-6, IL-10, IL-12/23 (p40), IL-8 and TNF-α were measuredusing Milliplex MAP Kit Non-Human Primate Cytokine Magnetic Beads Panel(Merck).

Result

The results of the cytokine measurements are shown using a relativevalue in case that the value of the reference compound (Compound 119) isevaluate as 100%. All compounds strongly induced the production of Th1cytokines in simian peripheral blood mononuclear cells. On the otherhand, they could hardly induce the production of Th2 cytokines. Th1/Th2ratio was 1 or more (Table 5).

TABLE 5 Cytokine profile when the oligonucleotide of the presentinvention is used. Seq. IFN- IL-12(p40) TNF- Th1/ No. γ NK, IFN-g α IL-6IL-4 IL-8 IL-10 Th2 119 100% 100% 100% 100% 100% 100% 100% 1 4 120 72 4556 L 245 22 5.40 5 — — — — — — — — 9 141 52 43 55 L 108 46 3.07 10 40 5823 54 L 72 47 0.86 20 323 58 39 43 L 130 17 18.76 22 160 76 42 59 L 13636 4.41 23 119 81 36 52 L 159 27 4.48 29 — — — — — — — — 30 — — — — — —— — 31 — — — — — — — — 39 95 56 27 46 L 65 30 3.11 53 434 120  100  71 L165 34 12.80 54 339 63 110  49 L 308 15 22.50 55 59 43 49 54 L 128 232.59 59 50 52 38 29 L 137 19 2.58 64 104 63 55 34 L 55 28 3.66 65 378 6763 47 L 110 26 14.29 66 142 58 52 33 L 172 21 6.77 67 589 69 74 53 L 13625 23.22 68 487 85 103  70 L 294 28 17.23 69 288 81 76 36 L 169 15 19.1670 164 63 74 57 L 125 26 6.25 71 213 67 102  44 L 326 15 14.56 74 133 5637 64 L 90 56 2.36 75 338 58 24 27 2 80  7 48.39 76 102 34 24 42 L 13930 3.44 77 51 51 26 28 L 127  9 5.46 78 147 117  161  135  L 1194 483.09 81 — — — — — — — — 84 115 53 58 64 L 223 39 2.98

L indicates that the value of measurement is below the detection limit.

Working Example 4

Efficacy Evaluation Test of Chiral Nucleic Acid Adjuvant Using AllergicRhinitis Model Mouse

Experimental Procedure

Preparation of the Model Mouse

The OVA and ALUM or the compounds of the present invention and mixturesof pertussis toxin were administered to 7 weeks-old BALB/cAnNCrlCrljintraperitoneally. This administration was referred to a firstsensitization. After 5 days, the OVA solution was subcutaneouslyadministered as an additional sensitization. As described above, thesystemic sensitization using OVA was performed. During 4 days after the18 days of the initial sensitization, the OVA solution was administratedin the nasal cavity of both sides of the mouse once a day using amicropipette. By this process, allergic symptoms was developed in themouse.

Administration of the Compounds

In order to investigate the effect on allergic symptoms onset ofoligonucleotide having a nucleotide sequence represented by SEQ ID NO:67 (Compound 67), 30 μg of the oligonucleotide was administrated to themouse subcutaneously 1 hour before the OVA initial sensitization orlocal sensitization.

The Number of Nose Scratching

In the fourth day of the OVA nasal administration (local sensitization),the number of nose scratching was counted for 1 hour immediately afterthe OVA nasal administration. Nose scratching was distinguished from thecleansing action and forelimb licking behavior, etc. Then, only theaction that the mouse scratched the nose in forelimb was counted as anasal scraping action. The results were shown in Table 6 and FIG. 1.

TABLE 6 Results of the measurement of the nose scratching (During onehour from just after the last nasal administration) Substances used forthe initial Test The number of nose scraching Avar- sensitization agent(in each sample) age 1 OVA + Alum Saline 44 57 73 28 50 148 66.67 2OVA + Alum Chlor- 1 12 21 29 12 14 14.83 phen- iramine 3 OVA + Com- Com-49 40 66 57 66 23 50.17 pound 67Sp pound 67Sp 4 OVA + Com- Com- 34 12867 39 57 33 59.67 pound 67Mix pound 67Mix

In Table 6, compound 67 mix indicates the oligonucleotide that part ofthe Sp-type was replaced with the Rp type in compound 67.

Measurement of OVA-Specific IgE in Mouse Serum

The concentration of OVA-specific IgE in blood collected after 5 days ofthe local sensitization was measured using Mouse Serum Anti-OVA IgEAntibody Assay Kit (Chondrex, Inc.). The kit was used in accordance withthe manufacturer's instructions, The results were shown in Table 7 andFIG. 2.

TABLE 7 Resuls of the measurement of OVA-specific IgE in mouse serumSubstances used for OVA-specific IgE the initial sensitization Testagent (Average ± SE) 1 OVA + Alum Saline 48.933 ± 14.458 2 OVA + AlumChlorpheniramine 27.984 ± 9.936  3 OVA + Compound 67Sp Compound 67Sp0.004 ± 0.003 4 OVA + Compound 67Mix Compound 67Rp 0.283 ± 0.107Result

Allergy symptoms was evaluated using the number of nose scratching as anindicator.

There is a tendency that the number of nose scraching in the groupadministrated compound 67 was reduced compared to that in the OVAadministration group. Sp stereoisomer of compound 67 strongly reducedthe number of nose scratching than stereoisomers mixture of compound 67(mix). On the other hand, in the compound 67 administrated group, theamount of OVA-specific IgE in serum was significantly reduced incomparison with the saline administrated group and the chlorpheniramineadministrated group. The chlorpheniramine administration group was usedas a positive control. In addition, the production of OVA-specific IgEwas completely prevented by administration of Sp stereoisomer of thecompound 67.

Working Example 5

Efficacy Evaluation Test of Chiral Nucleic Acid Adjuvant Using AllergicRhinitis Model Mouse

Experimental Procedure

Preparation of the Model Mouse (The Third Course)

The only OVA or the mixture of OVA and ALUM were administered to 7weeks-old BALB/cAnNCrlCrlj intraperitoneally. This administration wasreferred to a first sensitization. After 5 days, the liquid of OVA orthe mixture of the liquid of OVA and the compounds of the presentinvention (Sequence ID No. 67, Sp type stereoisomer) was subcutaneouslyadministered as systemic sensilization.

During 4 days after the 18 days of the initial sensitization, the OVAsolution was administrated in the nasal cavity of both sides of themouse once a day using a micropipette. By this process, allergicsymptoms was developed in the mouse.

Administration of the Compounds

In order to investigate the effect on allergic symptoms onset ofoligonucleotide having a nucleotide sequence represented by SEQ ID NO:67 (Compound 67), 10 μg of the oligonucleotide was administratedsubcutaneously or administrated in the nasal cavity of both sides of themouse at the additional sensitization, at 17th day from the firstsensitization or at the first date of the local sensitization.

Measurement of OVA Specific IgE in the Serums of Mouse

The OVA specific IgE in the serums of mouse was measured by means ofMouse Serum Anti-OVA IgE Antibody Assay Kit (Chondrex, Inc.) inaccordance with the instructions of the manufacturer using bloodobtained after 5 days from the local sensitization. The result is shownin the following table and FIG. 3.

TABLE 14 1st additional Group sensitization sensitization 1 OVA/SalineOVA + ALUM OVA 2 OVA + ALUM/OVA/ OVA + ALUM OVA chlorpheniramine 3 OVA +ALUM/OVA + OVA + ALUM OVA + 67Sp 67Sp 4 OVA + ALUM/OVA/ OVA + ALUM OVA67Sp (Day 17) 5 OVA + ALUM/OVA/ OVA + ALUM OVA OVA + 67Sp (Day 18) 6OVA + ALUM/OVA + OVA + ALUM OVA + 67Sp 67Sp/OVA + 67Sp (Day 18) OVA-IgEGroup Day 17 Day 18 Day20 (ng/mL) SE 1 — — comparison 50.89 17.09 2 — —chlorpheniramine 41.42 8.99 3 — — comparison 63.49 37.32 4 67Sp —comparison 25.61 5.61 5 — 67Sp comparison 60.62 9.73 6 — 67Sp comparison47.19 13.75

Count of Leukocytes in BALF, Bronchoalveolar Lavage Fluid.

BALF taken at autopsy (local sensitization after 5 days) was centrifuged(4 C, 800 g, 10 min). After removing the supernatant, the sediment isresuspend in 500 micro L of PBS. After mixing with Turk's solution,count is executed with the dilution rate was appropriately modifiedaccording to the number of cells. The results are shown in the followingtable and FIG. 4.

TABLE 15 Group 1 2 3 4 5 6 Neut. 1.75 2.33 0.42 0.42 0.25 0.08 SD 2.402.44 0.80 0.38 0.27 0.20 Eosi. 0.08 0.00 0.00 0.00 0.00 0.00 SD 0.200.00 0.00 0.00 0.00 0.00 Baso. 0.00 0.00 0.00 0.00 0.00 0.00 SD 0.000.00 0.00 0.00 0.00 0.00 Lymph. 0.83 1.00 0.25 0.25 0.67 0.92 SD 0.981.05 0.27 0.27 0.52 0.58 Mhg. 97.33 96.67 99.33 99.33 99.08 99.00 SD2.40 3.25 0.93 0.41 0.66 0.55

Neut. indicates the neutrophil, Eosi. indicate eosinophils, Baso.indicates the basophils, Lymph. Indicates lymphocytes, and Mhg.Indicates macrophages.

Results

The group that Sp stereoisomer of the compound 67 was administered oneday before the local sensitization (17th day from the firstsensitization) showed remarkable decrease in IgE in blood, which is anindication of allergy symptoms, compared with the group to whichchlorpheniramine was administered or the group OVA was administered. Thechlorpheniramine is a positive control.

Number of leukocytes in BALF, Bronchoalveolar Lavage Fluid, show thattendency of decreasing the number of neutrophil over the groups to whichSp-type stereoisomer of compound 67 was administered. However the groupto which chlorpheniramine was administered did not show such a tendency.

INDUSTRIAL APPLICABILITY

The present invention can be utilized in the field of pharmaceuticalindustry.

The invention claimed is:
 1. A method for treating an allergic reaction,comprising administering to a subject a composition comprising atherapeutically effective amount of the oligonucleotideT^(SP)C^(SP)GACGTT^(SP)T^(SP)T^(SP)GACGTT^(SP)T^(SP)T^(SP)GACG^(SP)T^(SP)T,wherein each ^(SP) represents an S type phosphorothioate linkage.
 2. Themethod as claimed in claim 1, further comprising administering ananti-allergic agent.
 3. A method for treating an allergic rhinitis,comprising administering to a subject a composition comprising atherapeutically effective amount of the oligonucleotideT^(SP)C^(SP)GACGTT^(SP)T^(SP)T^(SP)GACGTT^(SP)T^(SP)T^(SP)GACG^(SP)T^(SP)Twherein each ^(SP) represents an S type phosphorothioate linkage.